Abstract
Bcl-2 is often upregulated in cancers to neutralize the BH3-only protein Bim at the mitochondria. BH3 mimetics (e.g. ABT-199 (venetoclax)) kill cancer cells by targeting Bcl-2’s hydrophobic cleft and disrupting Bcl-2/Bim complexes. Some cancers with elevated Bcl-2 display poor responses towards BH3 mimetics, suggesting an additional function for anti-apoptotic Bcl-2 in these cancers. Indeed, Bcl-2 via its BH4 domain prevents cytotoxic Ca2+ release from the endoplasmic reticulum (ER) by directly inhibiting the inositol 1,4,5-trisphosphate receptor (IP3R). The cell-permeable Bcl-2/IP3R disruptor-2 (BIRD-2) peptide can kill these Bcl-2-dependent cancers by targeting Bcl-2’s BH4 domain, unleashing pro-apoptotic Ca2+-release events. We compared eight “primed to death” diffuse large B-cell lymphoma cell lines (DLBCL) for their apoptotic sensitivity towards BIRD-2 and venetoclax. By determining their IC50 using cytometric cell-death analysis, we discovered a reciprocal sensitivity towards venetoclax versus BIRD-2. Using immunoblotting, we quantified the expression levels of IP3R2 and Bim in DLBCL cell lysates, revealing that BIRD-2 sensitivity correlated with IP3R2 levels but not with Bim levels. Moreover, the requirement of intracellular Ca2+ for BIRD-2- versus venetoclax-induced cell death was different. Indeed, BAPTA-AM suppressed BIRD-2-induced cell death, but promoted venetoclax-induced cell death in DLBCL cells. Finally, compared to single-agent treatments, combining BIRD-2 with venetoclax synergistically enhanced cell-death induction, correlating with a Ca2+-dependent upregulation of Bim after BIRD-2 treatment. Our findings suggest that some cancer cells require Bcl-2 proteins at the mitochondria, preventing Bax activation via its hydrophobic cleft, while others require Bcl-2 proteins at the ER, preventing cytotoxic Ca2+-signaling events via its BH4 domain.
Highlights
Diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL) are amongst the most prevalent lymphoproliferative malignancies, characterized by an aggressive or indolent clinical behavior, respectively
We compared eight “primed to death” diffuse large B-cell lymphoma cell lines (DLBCL) for their apoptotic sensitivity towards Bcl-2/IP3R disruptor-2 (BIRD-2) and venetoclax. By determining their IC50 using cytometric cell-death analysis, we discovered a reciprocal sensitivity towards venetoclax versus BIRD-2
A collection of cancer cell lines mainly composed of germinal center DLBCL cells, which are highly dependent on Bcl-2 to survive the continuous and permanent death signaling, was used in the present study
Summary
Diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL) are amongst the most prevalent lymphoproliferative malignancies, characterized by an aggressive or indolent clinical behavior, respectively. A significant proportion of patients (29% of CD20-positive B-cell malignancies [1]) experiences www.impactjournals.com/oncotarget side effects or relapse after rituximab® treatment due to tumorescape mechanisms like downregulation of CD20 protein expression [2] Another important survival strategy of these B-cell malignancies is their upregulation of anti-apoptotic Bcl-2 proteins. Insights into the binding of Bcl-2 with the pro-apoptotic members have spurred the development of the BH3 mimetics These molecules mimic the BH3 domain of Bad, a sensitizer BH3-only protein thereby disrupting Bcl-2/Bim complexes and releasing Bim from the hydrophobic cleft of Bcl-2, which results in Bax/Bak-mediated apoptosis in cancer cells but not in healthy cells [8, 9]. Patients suffering from these poorly-primed cancers display poor responses to conventional chemotherapeutic drugs and seem hard to treat [14]
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